T cell specific cDNA clone

ABSTRACT

The invention provides a nucleic acid having a sequence which encodes a polypeptide that is at least part of a T cell antigen receptor. This encoded sequence is about 936 nucleotides in length and preferably is a human T cell antigen receptor. The nucleic acid sequence of one embodiment of the invention is shown in FIG. 3. 
     The nucleic acid sequence may be used as a probe to determine whether an unknown cell, e.g., a tumor cell, is a T cell. 
     Polypeptides encoded by the nucleic acid sequence include about 312 amino acids and are at least part of a T cell antigen receptor. They include at least one sequence which over 21 contiguous amino acids has greater than about 35% homology with mouse and human immunoglobin λ light chains. 
     Antibody to the polypeptide may be prepared and used to identify T cell antigen receptor and to determine whether an unknown cell, e.g., a tumor cell, is a T cell.

This is a division of application Ser. No. 06/577,526, filed Feb. 6,1984, now U.S. Pat. No. 4,713,332.

T (thymus derived) cells, like B cells, recognize specific antigens.This recognition is essential for activation of T cells, which have atleast two major functions in the immune system. They kill cells thatappear foreign, such as cells that have been infected with viruses andcarry viral antigens and they regulate other immune responses, includingantibody production by B cells (H. R. Green et al., (1983) Ann. Rev.Immunol. 1, 439-461; P. C. Kung et al., (1983) Intl. J. Dermatol. 22,67-76).

Antigen recognition is thought to be mediated through a receptor on Tcells. Molecular and biochemical characterization of the T cell antigenreceptor should clear up many of the mysteries concerning T cellrecognition of antigen and should help in attaining a betterunderstanding of the myriad interactions between the immune cells andtheir targets, and consequently, a better understanding of theregulation of immune responses.

In spite of extensive efforts, progress in characterizing the T cellantigen receptor during the past two decades has been slow (J. J.Marchalonis, (1982) Immunol. Today 3, 10-12; M. Kronenberg et al.,(1983) Cell. 34, 327-329). Recently several groups of investigatorshave, however, produced murine monoclonal antibodies that may recognizeT cell surface proteins with the predicted characteristics of theantigen receptor (J. L. Marx, (1983) Science 221, 444-446). Theseantibodies were produced by immunizing mice with cell lines of cloned Tcells; and the antibodies react with only the cells used to elicit theantibody production. Immunochemical studies in man (S. C. Meuer et al.,(1983) J. Exp. Med. 157, 705-719; S. C. Meuer et al., (1983) J. Exp.Med. 158, 988-993; R. D. Bigler et al., (1983) J. Exp. Med. 158,1000-1005; A. Oreste et al., (1983) Cell. 34, 717-726; J. Kappler etal., (1983) Cell. 35, 295-302; S. C. Meuer et al., (1983) Science 222,1239-1242) and in rodents (K. Haskins et al., (1983) J. Exp. Med. 157,1149-1161; P. Marrack et al., (1983) J. Exp. Med. 158, 1635-1646; B. W.McIntyre and J. P. Allison, (1983) Cell 34, 739-746; J. Kappler et al.,(1983) Cell. 34, 727-734) suggest that the putative T cell antigenreceptor is a molecule composed of two different polypeptide chains withmolecular weights of approximately 40,000 and 45,000 daltons,respectively. Under nonreducing conditions the T cell receptor moleculeis a disulfide-linked heterodimer with a molecular weight of 85,000 to90,000 daltons. Peptide mapping analysis in the murine system suggeststhat the T cell receptor protein contains a variable and a constantregion. However, these studies have provided no information concerningthe protein or nucleotide sequences.

Recently, there have appeared several reports that Stephen Hedrick atthe National Institutes of Allergy and Infectious Diseases (NIAID) andMark Davis, until recently with NIAID and now at Stanford MedicalSchool, have prepared cDNA from T cell mRNA which may encode a T cellreceptor protein (J. L. Marx, (1983) Science 221, 1278-1279; J.Berzofsky, (1983) Immunol. Today 4, 299-301). These reports suggest thatthe encoded protein may contain features in common with the variable andconstant regions of immunoglobulin chains. These reports, however, donot contain information concerning: (a) the amino acid sequence of theputative T cell receptor protein; (b) the precise size or nucleotidesequence of the cDNA prepared; (c) the origin of the T cells studied(presumably murine cells); (d) the experimental protocols followed; and(e) data concerning the cDNA or the protein. Therefore, these reportswould not enable one of ordinary skill in the art to reproduce orconfirm the conjectured results. In addition, there are no suggestionsconcerning possible uses of cDNA encoding the receptor protein or of thereceptor protein.

This invention relates to the isolation and detailed molecularcharacterization of a human T cell antigen receptor protein and the genewhich encodes it. The invention also concerns a method of isolating andscreening for the receptor gene, the nucleotide sequence of the gene,the amino acid sequence of the protein encoded by the gene and uses ofthe nucleotide sequence and the protein including the determinationwhether an unknown cell is a T cell.

The present invention provides a cloned cDNA which is specific for humanT lymphoid cells. The message was found to be expressed in human andmouse T lymphoblasts, thymocytes and phytohaemmaglutinin stimulated Tlymphocytes. Analysis of the amino acid sequence deduced from thenucleotide sequence indicates that its size and the relative positionsof the cysteine residues are similar to the light chain of mouse andhuman immunoglobulin molecules. The nucleotide sequence of the cDNA ofthe invention contains a stretch of extensive homology to the variable,joining and constant regions of the light chain proteins in both humanand mouse immunoglobulin. These features indicate that the cDNA clonecorresponds to a message that specifies part of the human T cellreceptor.

A particular description of the invention follows with reference beingmade to the drawings in which:

FIGS. 1a and 1b show a Northern blot examination of T and non-T cell RNAfor hybridization to clone YT35;

FIG. 2 is a SDS-polyacrylamide gel electrophoresis of the in vitrotranslation products of mRNA that hybridizes to clones YT35 and YT76;

FIG. 3 is a nucleotide and deduced amino acid sequence of a portion ofthe T cell-specific clone YT35 wherein T represents thymine nucleotide,G represents guanine nucleotide, A represents adenine nucleotide, and Crepresents cytosine nucleotide;

FIGS. 4a-d show a diagonal dot matrix comparison of the deduced aminoacid sequence of clone YT35 as shown in FIG. 3; and the amino acidsequence of mouse and human light chains; and

FIG. 5 is a direct comparison of the deduced amino acid sequence ofclone YT35 as shown in FIG. 3, and those of mouse and human λ lightchains.

The method whereby the cDNA clone was obtained utilized the humanleukemic T cell line MOLT-3 (Nagasawa, K. and Mak, T. W. (1982) CellImmunol 71, 390-403). This cell line is known to contain T cell specificantigens. Messenger RNA was isolated from MOLT-3 cells byoligo-(dT)-cellulose column chromatography, and cDNA was synthesizedusing the method of Land et al ((1981) Nucleic Acid Res. 9, 2251-2266).Using mRNA from MOLT-3 cells, double stranded cDNA was generated andinserted into the Bgl II site of the vector pFP502EB5 (Clark, S. P. andMak, T. W. (1982) Nuclei Acid Res. 10, 3315-3330). After transfectioninto E. coli strain HBl01, 10,000 independent cDNA clones were obtained.A random survey of 25 of these cDNA clones indicated that the lengths ofthe cDNA inserts were between 0.5 and 1.7 kb.

In order to screen for cDNA expressed either exclusively orpreferentially in T cells, the cDNA clones were grouped on the basis oftheir relative levels of expression in MOLT-3 cells and the human B cellline HSC-58. Four of these clones, YT30, YT35, YT53 and YT76, werespecific for MOLT-3 cells by Northern gel analysis. A single message of1.3 kb was detected in MOLT-3 cells but not in HSC-58. To test if thismessage was specific for T cell lines in general, hybridization with RNAfrom the following cell lines were examined:

(a) T cell lines Jurkat and CEM(T)

(b) B cell lines RPM 1 1788(B) and RMP1 3638(B)

(c) cells from a patient with B cell chronic lymphocytic leukemia

(d) the erythroleukemic cell line K562 and one obtained from H. Messner,Ontario Cancer Institute

(e) normal bone marrow cells

(f) bladder tumor cell line MGHU-1.

From FIG. 1a, it can be seen that this 1.3 kb message was expressed inall three T cell lines examined but was not expressed in any non-T cellsor cell lines (arrow).

The expression of these messages in other human and mouse T and B cellswere also examined by Northern gel analysis. The results which aresummarized in FIG. 1b confirm the above result that sequencescomplementary to these cDNA clones were expressed in MOLT-3 T cells butnot in HSC-58 B cells. In addition, the clones were expressed in normalhuman thymocytes, phytohaemmaglutinin (PHA) stimulated human peripheralblood T cells, and the mouse T cell line RBL-5 (FIG. 1b).

These results indicate that the cDNA clones YT30, YT35, YT53 and YT76were derived from mRNA common in T cell lines.

In order to determine the size of the protein encoded by the mRNAcorresponding to the T cell specific cDNA clones, the in vitro hybridselection method of Parnes et al was used ((1981) Proc. Natl. Acad. Sci.USA 78, 2253-2257). Total mRNA from MOLT-3 was allowed to anneal toclone YT35 or YT76. After one day, the unattached mRNA was removed andthe hybridized mRNA was released under low ionic conditions andtranslated in vitro with a rabbit reticulocyte lysate. The proteinsynthesized after this hybrid selection method is shown in FIG. 2. The30,000 dalton protein (arrow) appeared to be coded for by the mRNAselected by the T cell specific cDNA clone, while the larger proteinhaving a molecular weight of about 45,000 daltons was intrinsic to thein vitro translation system.

The nucleotide sequence of the YT35 clone was determined using thedideoxy chain terminating inhibitor method of Sanger et al ((1977) Proc.Natl. Acad. Sci. 74, 5463-5467). A complete sequence of 1151 nucleotidesis shown in FIG. 3. Examination of this sequence reveals a long openreading frame with a TGA termination triplet at position 974. In thelower portion of FIG. 3 the positions of the methionine codons (barsbelow line) and termination codons (bars above line) of the three codingframes are shown.

A protein that initiates at the first ATG methionine codon in thisreading frame (position 38) has a deduced molecular weight of 34938daltons, which is in general agreement with the results of hybridselection and in vitro translation (FIG. 2). There are stretches ofnonpolar uncharged amino acids in this protein adjacent the N and Ctermini (overlining in FIG. 3). The possible sites of N-glycosylationare shown in FIG. 3 by underlining.

In order to determine if the deduced amino acid sequence of the T cellspecific clone is similar to any of the immunoglobulin or T cell-relatedprotein sequences, these proteins were surveyed by diagonal dot-matrixanalysis (FIG. 4). It was found that a long region of homology wasperceptible between the protein sequences deduced from clone YT35 and amouse λ light chain (FIG. 4a) and human λ and K light chains. Thishomology is more easily recognized when the background is reduced bylooking for >35% homology over 21 contiguous amino acids. The resultingplots (FIGS. 4b, c and d) show that there are two stretches with highhomology, one near the C-terminal side of the variable region and theother near the N-terminal side of the constant region. A directcomparison of these sequences is shown in FIG. 5. The cysteine residuesat positions 42, 111, 166 and 231 of YT35 are underlined. Identitybetween the sequences is shown with asterisks (*) and vertical lines (|)denote conservative changes. Gaps (-) were introduced to increase thematchup. To identify conservative changes, the amino acids were groupedas follows--Acidic: aspartic acid (D) and glutamic acid (E); basic:histidine (H), lysine (K) and arginine (R); uncharged polar: asparagine(N), glutamine (Q), serine (S) and threonine (T); and nonpolar: alanine(A), cysteine (C), phenylalanine (F), glycine (G), isoleucine (I),leucine (L), methionine (M), proline (P), valine (V), tryptophan (W) andtyrosine (Y). The highest homology that was detected in FIG. 4corresponds to the region surrounding the cysteine residues at positions111 and 166 in the YT35 sequence. The sequences are quite similar nearthe cysteine residue at positions 42 and 231 as well. FIG. 5 alsoemphasizes the homology that was detected along the entire YT35 proteinin FIG. 4a. A comparison of the deduced amino acid sequence of cloneYT35 to a human K light chain protein showed slightly lower, but easilydetectable, homology than to the λ light chains (not shown). Lesspronounced homology was found to a heavy chains of human or mouseimmunoglobulins, but no significant homology was found to mouse H-2,Thy-1 or human HLA. The overall homology along the stretch of 249 aminoacids that overlaps with the λ light chains is 33% identical to themouse λ light chain and 38% in the constant region. As well, thehomology increases to 59% and 58% respectively if one takes intoconsideration conservative changes. The respective homology to theconstant region of human λ light chain is similar, at 36% identity and61% with conservative changes. It is of interest to note that theprotein encoded by clone YT35 is equally similar to the human and mouselight chains, and that the similarity is highest where the human andmouse light chains are most conserved between themselves. Also, thehighest homology to the λ and K chains occurs at different positions(FIG. 4).

The foregoing results indicate two important findings. First, a human Tcell-specific cDNA clone has been isolated, and second, it has beendetermined that the protein, as deduced from the nucleotide sequence,resembles human and mouse immunoglobulin light chain molecules as beingsimilar in the relative locations of the cysteine residues, but moreimportantly, extensive homology to the variable, joining, and constantregions can be found. These structural features indicate to one ofordinary skill that the protein encoded by YT35 is part of the antigenreceptor that mediates specialized T lymphocyte function.

A nucleic acid sequence of at least about 936 nucleotides encodes apolypeptide which is at least part of a T cell antigen receptor, e.g., aprimate T cell antigen receptor, preferably a human T cell antigenreceptor. The nucleic acid may be DNA, e.g., cDNA, or RNA. One such DNAsequence is shown in part in FIG. 3. Other such DNA sequences contain atthe 5' end at least about a 174 base sequence identical to the sequenceshown in FIG. 3 at positions 973 to 800. One such RNA sequence is asequence complementary to that shown in FIG. 3. Other such RNA sequencescontain at the 3' end at least about a 174 base sequence complementaryto the sequence shown in FIG. 3 at positions 973 to 800.

The nucleic acid sequences may be used as probes if labeled with adetectable marker, e.g., a radioactive, fluorescent or biotinylatedmarker. Labeled probe nucleic acid sequences according to the inventionmay be used, inter alia, to determine whether an unknown cell, e.g., atumor cell, is a T cell.

A cDNA sequence according to the invention may be prepared as follows.Total messenger RNA (mRNA) is obtained from a T cell, e.g., a MOLT-3cell or a thymocyte and used to prepare cDNA complementary to the mRNA.The cDNA is inserted into an appropriate cloning vehicle, e.g., pBR322or pFP502EBS, which is then inserted into a suitable host, e.g., E.coli. The resulting host is cultured under appropriate conditionspermitting production of multiple copies of the cDNA, and the cDNA soproduced is recovered and screened to determine whether it is expressedonly in T cells.

Host cells containing a suitable cloning vehicle which includes cDNAencoding the T cell antigen receptor may be prepared and used to producethe receptor polypeptide in accordance with methods known to those ofordinary skill in the art.

The invention also concerns polypeptides encoded by the nucleic acidsequences including a polypeptide which is at least part of a T cellantigen receptor and contains at least about 312 amino acids as shown inFIG. 3. Other polypeptides of at least about 312 amino acids whichcontain at the C-terminus at least about 58 amino acids identical to thesequence shown in FIG. 3 at positions 255 to 312 are T cell receptorantigens. Such polypeptides include at least one sequence which over 21contiguous amino acids has greater than about 35% homology with mouseand human λ light chains, such homology being present with sequences inthe variable, joining and constant regions.

Antibodies to the polypeptides, e.g., monoclonal or serum-derivedantibodies, may be prepared using methods known to those skilled in theart. Such antibodies may be employed to detect the presence of T cellreceptor antigen and to determine whether an unknown cell, e.g., a tumorcell, is a T cell. To do so the sample to be tested is treated with theantibody under suitable conditions permitting formation of antigenantibody complex and a determination made whether complex is present.

We claim:
 1. A method of determining whether an unknown cell is a T cellwhich comprises recovering nucleic acid from the unknown cell,contacting the unknown nucleic acid with nucleic acid having a sequencecomprising at least about 936 nucleotides and encoding a polypeptidewhich is at least part of the beta chain of a human T cell antigenreceptor, which sequence is labelled with a detectable marker, saidcontacting being under suitable conditions permitting hybridization, anddetecting whether hybridization of the unknown nucleic acid with saidsequence has occurred.
 2. A method of claim 1, wherein the unknownnucleic acid is DNA.
 3. A method of claim 1, wherein the unknown cell isa tumor cell.
 4. An isolated polypeptide encoded by a nucleic acidsequence which polypeptide is at least part of the beta chain of a humanT cell antigen receptor and which nucleic acid comprises at least about936 nucleotides.
 5. An isolated polypeptide of claim 4, wherein thenucleic acid sequence is a cDNA.
 6. An isolated polypeptide which is atleast part of the beta chain of a human T cell antigen receptor, saidpolypeptide comprising at least about 312 amino acids.
 7. An isolatedpolypeptide of claim 6 which contains at the C-terminus at least about58 amino acids identical to the sequence shown in FIG. 3 at positions255 to
 312. 8. An isolated polypeptide of claim 6 which includes atleast one amino acid sequence which sequence over 21 contiguous aminoacids has greater then about 35% homology with mouse and humanimmunoglobulin λ light chains.
 9. An isolated polypeptide of claim 8,wherein homology is present with sequence present in the variable,joining and constant regions of the λ light chains.
 10. An isolatedpolypeptide of claim 6 which includes the entire sequence shown in FIG.3.
 11. An isolated monoclonal antibody directed to an antigenicdeterminant on the polypeptide of claim
 6. 12. An isolated antibody ofclaim 11, wherein the antigenic determinant is unique to T cell antigenreceptor.
 13. A method of determining whether an unknown cell is a Tcell which comprises contacting the unknown cell with an antibody ofclaim 12 under suitable conditions permitting formation ofantigen-antibody complex and detecting whether complex is present.
 14. Amethod of claim 13, wherein the unknown cell is a tumor cell.
 15. Amethod of detecting in a sample the presence of T cell receptor antigenwhich comprises contacting the sample with an antibody of claim 12 undersuitable conditions permitting formation of antigen-antibody complex anddetecting whether said complex is present.
 16. An isolated oligomer orpolymer fragment of the polypeptide of claim 6, which fragment isgreater than about 35% homologous to the variable region of the lightchain protein of human or mouse immunoglobulin.
 17. An isolated oligomeror polymer fragment of the polypeptide of claim 6, which fragment ishomologous to the joining region of the light chain protein of human ormouse immunoglobulin.
 18. An isolated oligomer or polymer fragment ofthe polypeptide of claim 6, which fragment is homologous to the constantregion of the light chain protein of human or mouse immunoglobulin. 19.An isolated oligomer or polymer fragment of the polypeptide of claim 6,which fragment is greater than about 35% homologous to the diversityregion of the light chain protein of human or mouse immumoglobulin. 20.An isolated monoclonal antibody directed to an antigenic determinant onthe polypeptide of claim 10.